Transfection complexes

ABSTRACT

The invention provides a peptide having at least 3 amino acids comprising an amino acid sequence selected from 
                             a) X 1 SM   [SEQ.ID.NO.: 1]                   b) LX 2 HK   [SEQ.ID.NO.: 2]                   c) PSGX 3 ARA   [SEQ.ID.NO.: 9]                   d) SX 4 RSMNF   [SEQ.ID.NO.: 16]                   e) LX 5 HKSMP   [SEQ.ID.NO.: 18]                       
in which X 1  is a basic amino acid residue, X 2  is Q or P, X 3  is A or T, X 4  is an acidic amino acid residue and X 5  is P or Q.
 
     The invention further provides non-viral cell-targeting vector complexes and methods associated therewith.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/891,790, filedAug. 13, 2007 now U.S. Pat. No. 7,704,969, in issue, which applicationis a continuation of U.S. Ser. No. 10/471,895, filed Jan. 30, 2004 (nowU.S. Pat. No. 7,256,043), which is a United States national filing under35 U.S.C. §371 of international (PCT) application No. PCT/GB02/01215,filed Mar. 14, 2002, designating the United States, and claimingpriority to GB 01 06 315.5, filed Mar. 14, 2001.

BACKGROUND OF THE INVENTION

The present invention relates to peptides for use in an improved methodof transfecting cells.

The term “transfection” is used herein to denote the introduction of anucleic acid into a cell. The nucleic acid may be of any origin, and therecipient cell may be prokaryotic or eukaryotic.

Gene therapy and gene vaccination are techniques that offer interestingpossibilities for the treatment and/or prophylaxis of a variety ofconditions, as does anti-sense therapy. Such techniques require theintroduction of a DNA of interest into target cells. The ability totransfer sufficient DNA to specific target cells remains one of the mainlimitations to the development of gene therapy, anti-sense therapy andgene vaccination. Both viral and non-viral DNA delivery systems havebeen proposed. In some cases RNA is used instead of DNA.

Receptor-mediated gene delivery is a non-viral method of gene transferthat exploits the physiological cellular process, receptor-mediatedendocytosis to internalise DNA. Examples include vectors targetedagainst insulin receptors, see for example, Rosenkranz et alExperimental Cell Research 199, 323-329 (1992), asialoglycoproteinreceptors, see for example, Wu & Wu, Journal of Biological Chemistry262, 4429-4432 (1987), Chowdhury et al Journal of Biological Chemistry268, 11265-11271 (1993), and transferrin receptors, see for example,Ciriel et al, Proc. Natl. Acad. Sci. USA 88, 8850-8854 (1991). Furtherexamples of vectors include monoclonal antibodies targeting receptors onneuroblastoma cells (Yano et al, 2000), folate conjugated to liposomes(Reddy & Low 2000, Reddy et al. 1999), galactose for targeting livercells (Han et al. 1999 Bettinger et al. 1999) and asialogylcoprotein,also for liver cells (Wu et al. 1991).

Receptor-mediated non-viral vectors have several advantages over viralvectors. In particular, they lack pathogenicity; they allow targetedgene delivery to specific cell types and they are not restricted in thesize of nucleic acid molecules that can be packaged. Gene expression isachieved only if the nucleic acid component of the complex is releasedintact from the endosome to the cytoplasm and then crosses the nuclearmembrane to access the nuclear transcription machinery. However,transfection efficiency is generally poor relative to viral vectorsowing to endosomal degradation of the nucleic acid component, failure ofthe nucleic acid to enter the nucleus and the exclusion of aggregateslarger than about 150 nm from clathrin coated vesicles.

Desirable properties of targeting ligands for vectors are that theyshould bind to cell-surface receptors with high affinity and specificityand mediate efficient vector internalisation. Short peptides haveparticular advantages as targeting ligands since they arestraightforward to synthesise in high purity and, importantly for invivo use, they have low immunogenic potential.

WO 98/54347 discloses a mixture comprising an integrin-bindingcomponent, a polycationic nucleic acid-binding component, and a lipidcomponent, and also discloses a complex comprising

-   (i) a nucleic acid, especially a nucleic acid encoding a sequence of    interest,-   (ii) an integrin-binding component,-   (iii) a polycationic nucleic acid-binding component, and-   (iv) a lipid component.

The complex is primarily an integrin-mediated transfection vector.

Integrins are a super-family of heterodimeric membrane proteinsconsisting of several different α and β subunits. They are important forattachment of cells to the extracellular matrix, cell-cell interactionsand signal transduction. Integrin-mediated internalisation proceeds by aphagocytic-like process allowing the internalisation of bacterial cellsone to two micrometers in diameter (Isberg, 1991). Targeting ofnon-viral vectors to integrins, therefore, has the potential totransfect cells in a process that mimics infection of cells by pathogensand avoids the size limitation imposed by clathrin-coated vesicles inreceptor-mediated endocytosis.

It is considered that the components described in WO 98/54347 associateelectrostatically to form the vector complex, the vector being of thelipopolyplex type. The vector complexes of WO 98/54347 are found totransfect a range of cell lines and primary cell cultures with highefficiency, with integrin specificity and with low toxicity. Forexample, vascular smooth muscle cells are transfected with 50%efficiency, endothelial cells with 30% efficiency and haematopoieticcells with 10% efficiency. Furthermore, in vivo transfection ofbronchial epithelium of rat lung and pig lung with an efficiencycomparable with that of an adenoviral vector has been demonstrated.

Vectors that utilise integrin receptors to mediate gene transfer havethe advantage that they target a large number of different types ofcells in the body as integrin receptors are relatively widespread. Insome circumstances, for example, in in vivo treatment, however, it maybe preferable to target recipient cells more specifically.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide improved vectorcomplexes with enhanced cell targeting properties. The present inventionis based on the development of synthetic targeting non-viral vectorcomplexes that carry a ligand that is more cell-type selective than theligands of the prior art.

Previous approaches to targeted non-viral vectors have included the useof antibodies to substances involved in cell-cell adhesion. For example,vectors including monoclonal antibodies that target receptors onneuroblastoma cells (Yano et al, 2000) are known. Further examples oftargeting systems have proposed galactose for targeting liver cells (Hanet al. 1999 Bettinger et al. 1999) and asialogylcoprotein, also forliver cells (Wu et al. 1991). However, such methods have been effectiveonly in limited circumstances. For example, antibodies have broadapplicability but they are time-consuming to produce and, by virtue oftheir size, are not as suitable for in vivo administration to anorganism as a small molecule ligand. Furthermore, the methods previouslydescribed do not allow targeting to a cell type for which a ligand isnot yet available.

In the development of effective targeting vectors it is useful forseveral different target-binding ligands to be available. Effectivetargeted transfection requires not only good targeting but alsoeffective transfer of the vector DNA to the nucleus of the target cell.Even if a ligand is effective in targeting and binding to a target cell,effective gene transfection does not always occur. The reasons for thatare, at present, not clear. Accordingly, there remains a degree ofunpredictability regarding whether a ligand that binds effectively to atarget cell will also bring about effective transfection. It istherefore desirable to have available a “pool” of ligands for anyparticular cell surface receptor from which an effective transfectionligand may be selected. Such selection may take place by means of a genetransfer assay using, for example, a reporter gene, or by any othersuitable means.

The invention is based on the identification of specific peptidesequences that bind to human airway epithelial (HAE) cells. Theidentified families of HAE cell surface receptor binding componentpeptide motifs mediate specific binding to HAE cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides peptide having consisting of orcomprising an amino acid sequence selected from

a) X¹SM; [SEQ.ID.NO.: 1] b) LX²HK; [SEQ.ID.NO.: 2] c) PSGX³ARA;[SEQ.ID.NO.: 9] d) SX⁴RSMNF; [SEQ.ID.NO.: 16] and e) LX⁵HKSMP,[SEQ.ID.NO.: 18]in which X¹ is a basic amino acid residue, X² is Q or P, X³ is A or T,X⁴ is an acidic amino acid residue and X⁵ is P or Q.

Preferably, the peptide of the invention consists of or comprises anamino acid sequence selected from

a) X¹SM; [SEQ.ID.NO.: 1] b) LX²HK; [SEQ.ID.NO.: 2] and c) PSGAARA,[SEQ.ID.NO.: 3]in which X¹ is a basic amino acid residue and X² is Q or P.

Preferably X¹ is K or R. Preferably X² is P. Preferably X³ is A.Preferably X⁴ is E or Q [SEQ.ID.No.::17]. More preferably X⁴ is E.Preferably X⁵ is P.

Preferably, a peptide of the invention comprises a sequence selectedfrom LQHKSMP [SEQ.ID.NO.:4], LPHKSMP [SEQ.ID.NO.:5], VKSMVTH[SEQ.ID.NO.:6], SERSMNF [SEQ.ID.NO.:7], VGLPHKF [SEQ.ID.NO.:8], YGLPHKF[SEQ.ID.NO.:19], PSGAARA [SEQ.ID.NO.:3], SQRSMNF [SEQ.ID.NO.:36] andPSGTARA [SEQ.ID.NO.:38]. Most preferably, the peptide comprises asequence selected from LQHKSMP [SEQ.ID.NO.:4], and LPHKSMP[SEQ.ID.NO.:5].

A peptide of the invention may be up to 20 amino acids in length, or maybe longer. A peptide of the invention generally has at least 5 aminoacids but may have perhaps fewer. Generally, a peptide of the inventionhas any number of amino acids from 6 to 20 inclusive. The peptide mayhave 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 aminoacids. Generally, it is preferred for a peptide of the invention to have15 amino acids or fewer. For example, a peptide of the invention mayhave 12 amino acids or fewer. Preferably a peptide of the inventionaccording to the invention has 10 amino acids or fewer. Generally, it ispreferred for a peptide of the invention to have 5 or more amino acids.For example, a peptide of the invention may have 6 or more amino acids.For example a peptide of the invention has 7 amino acids. In the case ofa peptide comprising amino acid sequence c) above, the minimum size is 7amino acids.

Preferably, a peptide of the invention is such that X¹ is K or R or X²is Q or P.

A peptide of the invention may comprise a cyclic region. Preferably, themotif of the invention is flanked by two or more cysteine residues thatare capable of forming one or more disulphide bond(s). For example, apeptide of the invention may be “peptide P′” (CLPHKSMPC [SEQ.ID.NO.:10])or “peptide Q′” (CLQHKSMPC [SEQ.ID.NO.:11]).

The peptides of the invention find use in HAE cell targeted non-viraltransfection vector complexes. They are also useful in targeted viraltransfection vectors.

The peptide is preferably linked to a polycationic nucleic acid bindingcomponent. The polycationic nucleic acid binding component may be anypolycationic molecule suitable for binding a nucleic acid.

For example, it may be polyethylenimine. Polyethylenimine (PEI) is anon-toxic, cross linked cationic polymer with gene delivery potential(Proc. Natl. Acad. Sci., 1995, 92, 7297-7301). For example, the peptidemay be linked to the PEI structure via a disulphide bridge using methodsknown in the art (for example, Gene Therapy, 1999, 6, 138-145).Polyethylenimine is obtainable from Fluka (800 kDa) or from Sigma (50kDa) or alternatively pre-diluted for transfection purposes fromPolyPlus-transfection (Illkirch, France). Typically, PEI is mostefficient when in a 9 fold excess over DNA (the excess ratio beingcalculated as PEI nitrogen:DNA phosphate) and at pH 5-8. Such parametersmay optimised in a manner familiar to the person skilled in the art.

Another example of a nucleic acid-binding polycationic molecule is anoligopeptide comprising one or more cationic amino acids. Such aoligopeptide may, for example, be an oligo-lysine molecule having from 5to 25 lysine moieties, preferably having from 10 to 20 lysine moieties,for example 16 lysine moieties, an oligo-histidine molecule, or anoligo-arginine molecule or a combined oligomer comprising anycombination of histidine, arginine and lysine residues and having atotal of from 5 to 25 residues, preferably having from 10 to 20residues, for example 16 residues.

The peptide may be attached to the polycationic nucleic acid bindingcomponent via a spacer.

A spacer element is generally a peptide, that is to say, it comprisesamino acid residues. The amino acids may be naturally occurring ornon-naturally occurring. They may have L- or D-configuration. A spacermay have two or more amino acids. It may, for example, comprise three ormore amino acids, for example, four or more, for example, five or more,for example, up to ten amino acids or more. The amino acids may be thesame or different, but the use of multiple lysine residues (or othercationic amino acids suitable for use in the polycationic nucleicacid-binding component of a vector complex) should be avoided in thespacer as oligolysine sequences have activity as a polycationic nucleicacid-binding component of a vector complex of the present invention.

The spacer may be, for example, the dipeptide glycine-glycine (GG) orglycine-alanine (GA). Generally it is preferable that the spacer islonger and/or more hydrophobic than the dipeptide spacers GG and GA.

The spacer may be more hydrophobic than the dipeptides GG and GA. Forexample, amino acids that are more hydrophobic than glycine and alaninemay be used. Examples of hydrophobic amino acids are well known andinclude ε-amino hexanoic acid.

A spacer may be either longer or more hydrophobic than the dipeptides GGand GA, or it may be both longer and more hydrophobic. An example of thelatter type of spacer is XSXGA, wherein S=serine, G=glycine, A=alanineand X=ε-amino hexanoic acid. This spacer is highly hydrophobic.

The invention further provides a peptide derivative of formula A-B-Cwherein

-   -   A is a polycationic nucleic acid-binding component,    -   B is a spacer element, and    -   C is a peptide as described above.

Polycationic nucleic acid-binding component A may be any polycationicnucleic acid-binding component as described above. Spacer element B maybe any of the spacer elements described above.

The invention further provides a non-viral transfection complexcomprising:

-   -   (i) a nucleic acid,    -   (ii) a lipid component,    -   (iii) a polycationic nucleic acid-binding component, and    -   (iv) a cell surface receptor binding component, comprising a        peptide as described above.

The cell surface receptor binding component may have the featuresdescribed above in relation to the peptides of the invention.

The cell surface receptor binding component peptides were identified byselection from a peptide library of random 7-mers (peptides having sevenamino acid residues) and random 12-mers (peptides having twelve aminoacid residues) displayed on filamentous phage particles. Resultsobtained using the random 7-mer library were better than those using therandom 12-mer peptide library. The reasons for the difference inperformance of the seven and twelve amino acid library are not known atpresent. It is possible that the larger amino acid insert in the phagecoat protein reduces the viability of the phage and/or that theadditional protein synthesis requirement places too great a burden onthe E. coli bacteria. Alternatively, or in addition, impurities in ordefects of the 12-mer library may have adversely affected the outcome ofthe experiments with that library. It appears, however, that smallerpeptides, for example heptameric peptides are preferred. Accordingly,the peptide of the invention preferably has 4 to 11 amino acids, morepreferably 4 to 10 amino acids, for example 7 amino acids.

The 7-mer library used was a C7C library (i.e. random 7-mer peptidesflanked by cysteine residues) obtained from New England Biolabs Inc. The12-mer library used was also obtained from New England Biolabs Inc.

As indicated above, the HAE cell surface receptor binding peptides ofthe invention were identified by selection from a phage display librarycomprising random peptide sequences seven residues in length flanked bycysteine residues to allow cyclisation. Such selection procedures aregenerally known. According to such procedures, suspensions of phage areincubated with target cells. Unbound phage are then washed away and,subsequently, bound phage are extracted either by washing the remainingcells with a low pH buffer or by lysing the cells. E. coli are theninfected with released phage and a preparation of first round phage isobtained. The cycle is performed repeatedly, for example three timesand, in order to enrich for targeting phage, the stringency conditionsmay be increased in the later rounds of selection, for example byincreasing the number of wash steps, introducing a low pH wash prior toelution and preselecting with wells coated with medium blocker.

Following selection by successive rounds of phage amplification, it hasbeen found that phage with high affinity for HAE cells may be selectedfurther by whole cell ELISA using plated HAE cells.

Following incubation of the phage with the HAE cells, the cells arewashed and retained phage may then be detected by immunostaining. Cellspecificity is assessed by comparing phage binding to target cells withphage binding to the wells on which the cells were plated and with phagebinding to NIH 3T3 fibroblast control cells.

Using the whole cell ELISA (Enzyme-Linked ImmunoSorbent Assay) assaydescribed above, high affinity and high specificity binding peptideswere identified. The cells to which high affinity phage were bound werelysed to release the bound phage particles. The phage DNA was isolatedand sequenced.

The amino acid sequences of clones obtained from cell lysis eluted C7Cphage in a first experiment are shown in Table 1a.

TABLE 1a Sequence Clone frequency SEQ.ID LQHKSMP 3 4 LPHKSMP 1 5 YGLPHKF1 19 SERSMNF 3 7 VKSMVTH 2 6 PSGAARA 2 3

The amino acid sequences of clones obtained from cell lysis eluted C7Cphage in a second experiment are shown in Table 1b.

TABLE 1b Sequence Clone Frequency SEQ.ID.NO. SERSMNF 18 7 YGLPHKF 12 19PSGAARA 9 3 LQHKSMP 3 4 VKSMVTH 3 6 SQRSMNF 2 36 QPLRHHQ 2 37 LPHKSMP 15 PSGTARA 1 38 KQRPAWL 1 39 IPMNAPW 1 40 SLPFARN 1 41 GPARISF 1 42MGLPLRF 1 43

The 56 sequenced clones from the third round of panning of HAEo− in thesecond experiment were represented by the 14 sequences shown in Table1b, with some sequences being represented by multiple phage clones. Thesequences shown were each flanked by two cysteine residues in the phageand are thus constrained in a loop formation by disulphide bonds betweenthem. For the avoidance of doubt, all of the sequences in Tables 1a and1b form part of the present invention

An analysis of the motifs found in the positive clone amino acidsequences of Table 1a (the first experiment) is shown in Table 2a.

TABLE 2a SEQ. Clone Motif Motif Sequence ID. frequency frequency KSM/RSMLQHKSMP 4 3 9 LPHKSMP 5 1 VKSMVTH 6 2 SERSMNF 7 3 LXHK LQHKSMP 4 3 5LPHKSMP 5 1 YGLPHKF 19 1 LXHKSMP LQHKSMP 4 3 4 (SEQ ID NO: 18) LPHKSMP 51 PSGAARA* PSGAARA 3 2 2 *PSGAARA (SEQ ID NO: 3) is not a motif, but arepeated clone in the first experiment not containing any motifs alreadyidentified.

An analysis of the motifs found in the positive clone amino acidsequences of Table 1b (the second experiment) is shown in Table 2b.

TABLE 2b SEQ. Clone Motif Motif Sequence ID. frequency frequency KSM/RSM SERSMNF 7 18 27  SQRSMNF 36 2   VKSMVTH 6 3  LQHKSMP 4 3  LPHKSMP 5 1SXRSMNF SERSMNF 7 18 20 (SEQ ID NO: 16) SQRSMNF 36 2 LXHK   LQHKSMP 4 316 (SEQ ID NO: 12)   LPHKSMP 5 1 YGLPHKF 19 12 PSGXAPA PSGAARA 3 9 10(SEQ ID NO: 9) PSGTARA 38 1 LXHKSMP LPHKSMP 5 3 4 (SEQ ID NO: 18)LQHKSMP 4 1

The sequences found in the first experiment (Table 1a) were compared andranked for their binding strength by ELISA using a range of phage titres(Table 3). In Table 3, the sequences are ranked in order of bindingaffinity to HAE cells. It was found that the sequence LPHKSMP (SEQ IDNO: 5) (“Peptide P”) had the highest binding affinity.

TABLE 3 Clone Sequence SEQ. ID frequency Motifs LPHKSMP 5 1 LXHK (SEQ IDNO: 2), LXHKSMP (SEQ ID NO: 18), KSM LQHKSMP 4 3 LXHK (SEQ ID NO: 2),LXHKSMP (SEQ ID NO: 18), KSM YGLPHKF 19 1 LXHK (SEQ ID NO: 2) VKSMVTH 62 KSM PSGAARA 3 2 PSGAARA (SEQ ID NO: 9) SERSMNF 7 3 RSM

From the Tables it may be seen that the motifs KSM/RSM and LXHK werepresent in several of the clones. This strongly suggests that thosemotifs are important for HAE cell surface binding. It is at present notknown to which HAE receptor(s) the sequences bind. The various motifsmay target the same receptor or they may target different receptors.

Good binding indicates a high affinity interaction and/or the binding ofa cell surface receptor molecule present in high numbers on the cellsurface. The LPHK version of the LXHK motif provides better binding thanthe LQHK version and the KSM version of the XSM motif provides betterbinding than the RSM version. The LXHK motif and the KSM motif arefrequently found together. This may be due to a cooperative effect,possibly due to the motifs binding to two cell surface receptormolecules.

Although the peptide sequences of the invention were identified usingHAE cells, their utility is not limited to use with HAE cells. Thereceptors to which the peptides bind may be expressed in other celltypes. Cell types with which peptides of the invention may be used maybe identified by any suitable screening procedure.

The transfection properties the vector complexes of the invention wereinvestigated in HAE cell transfection experiments as described below.

Non-viral transfection vector complexes incorporating the identifiedsequences were prepared. Peptides were synthesised using standard solidphase synthetic chemistry and a sixteen-lysine tail was added. The mostfrequently occurring peptides were chosen for synthesis, with peptideLPHKSMP chosen because it contains three motifs. Each peptide wasassigned a single letter name. The peptides chosen for synthesis areshown in Table 4.

TABLE 4 Clone Peptide Sequence SEQ. ID. frequency Motifs E SERSMNF 7 18RSM, SXRSMNF (SEQ ID NO: 16) Y YGLPHKF 19 12 LXHK (SEQ ID NO: 2) GPSGAARA 3 9 PSGXARA (SEQ ID NO: 9) V VKSMVTH 6 3 KSM Q LQHKSMP 4 3 LXHK(SEQ ID NO: 2), LXHKSMP (SEQ ID NO: 18), KSM P LPHKSMP 5 1 LXHK (SEQ IDNO: 2), LXHKSMP (SEQ ID NO: 18), KSM (Where X = any amino acid)

Luciferase reporter gene DNA was used as the transfection DNA.Transfection complexes were made by mixing the components in theorder 1) lipid, then 2) peptide and, finally 3) DNA, followed bydilution. The vector complex suspension was applied to HAE cells andcontrol cells. Vector complexes incorporating peptide Q([K]₁₆-GACLQHKSMPCG [SEQ.ID.NO.:12]) and vector complexes incorporatingpeptide P ([K]₁₆-GACLPHKSMPCG [SEQ.ID.NO.:13]) were synthesised andcompared with vector complexes incorporating peptide S([K]₁₆-GACYKHPGFLCG] [SEQ.ID.NO.:14]) which is a control peptide havingthe same amino acid constituents as peptide P but in a randomised order(the “scrambled control”), Peptide 12 ([K]₁₆-XSXGACRRETAWACG[SEQ.ID.NO.:15]), a targeting peptide known to bind to alpha 5 beta 1integrins and Peptide K ([K]₁₆) a DNA binding moiety with no targetingligand attached.

Transfections of HAE cells and 3T3 cells were performed in 96 wellplates containing 20,000 cells plated 24 hours earlier. In thetransfection vector complex, peptide to DNA charge ratios (+/−) wereused at 1.5:1, 3:1 and 7:1. At physiological pH, DNA carries negativecharge and the polycationic-nucleic acid binding component carriespositive charge. The “charge ratio” is accordingly the ratio of thecharges of the two components in the complex. The lipid component wasmaintained at a constant proportion, by weight, relative to DNA of0.75:1. The results of the transfection experiments are shown in FIG. 3.

At a 7:1 charge ratio, the transfection efficiency of vector complexescontaining peptide P was five-fold higher than the next best peptide,peptide 12 at a 3:1 charge ratio. Peptide P was 150-fold better thanpeptide S (the scrambled control) at the charge ratio of 7:1 indicatingthat the transfection efficiency was receptor specific. Vector complexescontaining peptide P were almost nine-fold more efficient thosecontaining peptide K, again indicating receptor specificity. The factthat vector complexes containing peptide K performed better in the assaythan vector complexes containing peptide S suggests that sterichindrance by the scrambled motif in peptide S may play a role.

Despite the similar HAE cell surface binding properties of peptide P andpeptide Q (See FIG. 2), peptide P performed significantly better thanpeptide Q in the transfection assay. This result suggests that bindingproperties alone are not sufficient to achieve high efficiency oftransfection.

The HAE cell surface receptor binding peptide component for use in thevector complex of the invention may be synthesised using standard solidphase peptide synthesis methods.

The identity of the molecules bound by the peptides used intransfections was explored by carrying out a BLAST search (Tables 5a and5b). Homologies were found to several molecules of interest which maybind molecules present on the surface of epithelial cells in the lung.Pathogen peptides with homology with the peptides of the invention areshown in Table 5a, whilst cell adhesion molecules with homology with thepeptides of the invention are shown in Table 5b.

TABLE 5a Peptide Homology Protein Pathogen Receptor LPHKSMP/ LHKSMGlycoprotein B Human Cell surface LQHKSMP herpesvirus heparan sulphateSXRSMNF SDRSMN Capsid binding Human ICAM-1 or LDL protein VP2 rhinovirusreceptor family YGLPHKF YGLPHK Unknown Legionella Unknown epithelialpneumophila cell receptors VKSMVTH VKSMITQ Adhesin P1 Mycoplasma Cellsurface Pneumoniae sialoglycoproteins

TABLE 5b Peptide Homology Protein Species Receptor SXRSMNF SERSMNSelectin Rat Cell surface glycoproteins ERSMDF Laminin, alpha HumanExtracellular 5 matrix components including integrins LXHKSMP LPHKNMEpithelial Mouse/ Dimerises, also caderin rabbit binds integrin(ovumorulin) a-E, b-7

Epithelial cadherin is a molecule which is involved in cell-celladhesion and forms complexes with β-catenin. Human herpesvirusglycoprotein B binds cell surface heparan sulphate proteoglycans.Selectin binds cell surface glycoproteins. Laminin, alpha 5 is abasement membrane protein found in epithelium. The capsid bindingprotein VP2 of the rhinovirus binds ICAM-1 or the LDL receptor family ofmolecules in the upper respiratory tract. P-glycoprotein is a molecularpump molecule which is localised in the cell membrane, and coagulationfactor XII has been shown to bind cytokeratins on epithelial cells.

In so far as any motif or any peptide of the invention occurs in anaturally-occurring protein, the peptides of invention do not includesuch a naturally-occurring full-length protein. Generally, the peptidesof the invention are 100 or fewer amino acids in length; preferably thepeptides of the invention are 50 or fewer amino acids in length.Typically, they are of sizes described above.

The peptides of the invention finds utility in the study of conditionsinvolving the pathogens and the cell adhesion molecules given in Tables4a and 4b. They are also useful in the development of treatments forthose conditions.

The nucleic acid component may be obtained from natural sources, or maybe produced recombinantly or by chemical synthesis. It may be modified,for example, to comprise a molecule having a specific function, forexample, a nuclear targeting molecule. The nucleic acid may be DNA orRNA. DNA may be single stranded or double stranded. The nucleic acid maybe suitable for use in gene therapy, in gene vaccination or inanti-sense therapy. The nucleic acid may be or may relate to a gene thatis the target for particular gene therapy or may be a molecule that canfunction as a gene vaccine or as an anti-sense therapeutic agent. Thenucleic acid may be or correspond to a complete coding sequence or maybe part of a coding sequence.

Alternatively, the nucleic acid may encode a protein that iscommercially useful, for example industrially or scientifically useful,for example an enzyme; that is pharmaceutically useful, for example, aprotein that can be used therapeutically or prophylactically as amedicament or vaccine; or that is diagnostically useful, for example, anantigen for use in an ELISA. Host cells capable of producingcommercially useful proteins are sometimes called “cell factories”.

Appropriate transcriptional and translational control elements aregenerally provided. For gene therapy, the nucleic acid component isgenerally presented in the form of a nucleic acid insert in a plasmid orvector. In some cases, however, it is not necessary to incorporate thenucleic acid component in a vector in order to achieve expression. Forexample, gene vaccination and anti-sense therapy can be achieved using anaked nucleic acid.

The nucleic acid is generally DNA but RNA may be used in some cases, forexample, in cancer vaccination. The nucleic acid component may bereferred to below as the plasmid component or component “D”.

As indicated above, the polycationic nucleic acid-binding component isany polycation that is capable of binding to DNA or RNA. The polycationmay have any number of cationic monomers provided the ability to bind toDNA or RNA is retained. For example, from 3 to 100 cationic monomers maybe present, for example, from 10 to 20, for example from 14 to 18,especially about 16. An oligolysine is particularly preferred, forexample, having from 10 to 20 lysine residues, for example, from 13 to19, for example, from 14 to 18, for example, from 15 to 17 residues,especially 16 residues i.e. [K]₁₆, “K” denoting lysine.

A further preferred cationic polymer is polyethylenimine (Proc. Natl.Acad. Sci., 1995, 92, 7297-7301).

The polycationic DNA-binding or RNA-binding component may advantageouslybe linked or otherwise attached to the cell surface receptor-bindingcomponent. A combined cell surface receptor-bindingcomponent/polycationic DNA-binding or RNA-binding component may bereferred to below as component “I”. For example, a polycationicDNA-binding or RNA-binding component may be chemically bonded to a cellsurface receptor-binding compo-nent, for example, by a peptide bond inthe case of an oligolysine. The polycationic component may be linked atany position of the cell surface receptor-binding component. Preferredcombinations of cell surface receptor-binding component and polycationicDNA-binding or RNA-binding component are an oligolysine, especially[K]₁₆, linked via a peptide bond to a peptide, for example, a peptide asdescribed above. A further preferred combination of cell surfacereceptor-binding component and polycationic DNA-binding or RNA-bindingcomponent are a polyethylenimine linked via a covalent link to apeptide, for example, a peptide as described above. For example such acovalent link may be a disulphide bridge or a succinimidyl bridge.

The lipid component may be or may form a cationic liposome. The lipidcomponent may be or may comprise one or more lipids selected fromcationic lipids and lipids having membrane destabilising or fusogenicproperties, especially a combination of a cationic lipid and a lipidthat has membrane destabilising properties.

A preferred lipid component (“L”) is or comprises the neutral lipiddioleyl phosphatidylethanolamine, referred to herein as “DOPE”. DOPE hasmembrane destabilising properties sometimes referred to as “fusogenic”properties (Farhood et al. 1995). Other lipids, for example, neutrallipids, having membrane destabilising properties, especially membranedestabilising properties like those of DOPE may be used instead of or aswell as DOPE.

Other phospholipids having at least one long chain alkyl group, forexample, di(long alkyl chain)phospholipids may be used. The phospholipidmay comprise a phosphatidyl group, for example, aphosphatidylalkanolamine group, for example, a phosphatidyl-ethanolaminegroup.

A further preferred lipid component is or comprises the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, referredto herein as “DOTMA”. DOTMA has cationic properties. Other cationiclipids may be used in addition to or as an alternative to DOTMA, inparticular cationic lipids having similar properties to those of DOTMA.Such lipids are, for example, quaternary ammonium salts substituted bythree short chain alkyl groups, and one long chain alkyl group. Theshort chain alkyl groups may be the same or different, and may beselected from methyl and ethyl groups. At least one and up to three ofthe short chain alkyl group may be a methyl group. The long alkyl chaingroup may have a straight or branched chain, for example, a di(longchain alkyl)alkyl group.

Another preferred lipid component is or comprises the lipid2,3-dioleyloxy-N-[2-(spermidinecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoridoacetate,referred to herein as “DOSPA”. Analogous lipids may be used in additionto or as an alternative to DOSPA, in particular lipids having similarproperties to those of DOSPA. Such lipids have, for example, differentshort chain alkyl groups from those in DOSPA.

A preferred lipid component comprises DOPE and one or more other lipidcomponents, for example, as described above. Especially preferred is alipid component that comprises a mixture of DOPE and DOTMA. Suchmixtures form cationic liposomes. An equimolar mixture of DOPE and DOTMAis found to be particularly effective. Such a mixture is knowngenerically as “lipofectin” and is available commercially under the name“Lipofectin”. The term “lipofectin” is used herein generically to denotean equimolar mixture of DOPE and DOTMA. Other mixtures of lipids thatare cationic liposomes having similar properties to lipofectin may beused. Lipofectin is particularly useful as it is effective in all celltypes tested.

A further preferred lipid component comprises a mixture of DOPE andDOSPA. Such mixtures also form cationic liposomes. A mixture of DOPE andDOSPA in a ratio by weight 3:1 DOSPA:DOPE is particularly effective.Such a mixture, in membrane filtered water, is available commerciallyunder the name “Lipofectamine”. Mixtures comprising DOPE, DOTMA andDOSPA may be used, for example, mixtures of lipofectin andlipofectamine.

Other cationic lipids are available commercially, for example, DOTAP(Boehringer-Mannheim) and lipids in the Tfx range (Promega). DOTAP isN-[1-(2,3-diolyloxy)propyl]-N,N,N-tri-methylammonium methylsulphate. TheTfx reagents are mixtures of a synthetic cationic lipid[N,N,N′,N′-tetramethyl-N,N′-bis(2-hydroxyethyl)-2,3-di(oleoyloxy)-1,4-butanediammoniumiodide and DOPE. All the reagents contain the same amount of thecationic lipid component but contain different molar amounts of thefusogneic lipid, DOPE.

However, lipofectin and lipofectamine appear to be markedly moreeffective as the lipid component in LID vector complexes of the presentinvention than are DOTPA and Tfx agents.

The effectiveness of a putative cell surface receptor-binding component,polycationic DNA-binding or RNA-binding component, or of lipid componentor of any combination thereof may be determined readily using themethods described herein.

The efficiency of transfection using a transfection complex as describedabove as transfection vector is influenced by the ratio lipidcomponent:cell surface receptor-binding component:DNA or RNA. For anychosen combination of components for any particular type of cell to betransfected, the optimal ratios can be determined simply by admixing thecomponents in different ratios and measuring the transfection rate forthat cell type, for example, as described herein.

Lipofectin and lipofectamine appear to be particularly effective inenhancing transfection in the system described above. Lipofectin has theadvantage that only very small amounts are required. Any side effectsthat may occur are therefore minimised. A suitable weight ratio betweenthe lipid and the DNA components has been found to be 0.75:1. For anygiven transfection experiment, this ratio may be optimised using methodsknown in the art.

Cells that may be transfected by a transfection vector complexincorporating a peptide of the invention include, for example,endothelial or epithelial cells, for example, cells of the any part ofthe airway epithelium, including bronchial and lung epithelium, and thecorneal endothelium. The airway epithelium is an important target forgene therapy for cystic fibrosis and asthma.

A transfection vector complex as described above may be produced byadmixing components (i), (ii), (iii) and (iv).

Although the components may be admixed in any order, it is generallypreferable that the lipid component is not added last. In the case wherethere is a combined cell surface receptor-binding component/polycationicDNA-binding or RNA-binding component it is generally preferable tocombine the components in the following order: lipid component; combinedcell surface receptor-binding/polycationic DNA-binding or RNA-bindingcomponent; DNA or RNA component, for example, in the order: lipofectin,oligolysine-peptide component, DNA or RNA component.

A transfection mixture comprising a cell surface receptor-bindingcomponent, a polycationic nucleic acid-binding component, and a lipidcomponent may be used to produce a nucleic acid-containing transfectionvector complex as described above by the incorporation of a nucleic acidwith the mixture, for example, by admixture. Alternatively, thetransfection mixture may be used for the production of a vector complexwhich comprises, instead of the nucleic acid component, any othercomponent that is capable of binding to the polycationic nucleic-acidbinding component, for example, a protein.

The individual components of a transfection mixture of the invention areeach as described above in relation to the transfection vector complex.The preferred components, preferred combinations of components,preferred ratios of components and preferred order of mixing, both withregard to the mixture and to the production of a vector complex, are asdescribed above in relation to the transfection vector complex.

A transfection mixture preferably comprises an equimolar mixture of DOPEand DOTMA (lipofectin) as the lipid component and an oligolysine-peptideespecially a [K]₁₆-peptide as a combined cell surface receptor-bindingcomponent/nucleic acid-binding component. The preferred molar ratiolipofectine:oligolysine-peptide is 0.75:4.

The invention further provides a non-viral transfection complexcomprising:

-   -   (i) a nucleic acid,    -   (iii) a polycationic nucleic acid-binding component, and    -   (iv) a cell surface receptor binding component, comprising a        peptide as described above.

The cell surface receptor binding component may have the featuresdescribed above in relation to the peptides of the invention. Thenucleic acid component and the polycationic nucleic acid-bindingcomponent may be as described above in relation to the non-viraltransfection complex comprising (i), (ii), (iii) and (iv).

The effectiveness of a putative cell surface receptor-binding componentand polycationic DNA-binding or RNA-binding component may be determinedreadily using the methods described herein.

The efficiency of transfection using a transfection complex as describedabove as transfection vector is influenced by the ratio of cell surfacereceptor-binding component:polycationic nucleic acid-bindingcomponent:DNA or RNA. For any chosen combination of components for anyparticular type of cell to be transfected, the optimal ratios can bedetermined simply by admixing the components in different ratios andmeasuring the transfection rate for that cell type, for example, asdescribed herein.

Cells that may be transfected by a transfection vector complexincorporating a peptide of the invention include, for example,endothelial or epithelial cells, for example, cells of any part of theairway epithelium, including bronchial and lung epithelium, and thecorneal endothelium. The airway epithelium is an important target forgene therapy for cystic fibrosis and asthma.

A transfection vector complex as described above may be produced byadmixing components (i), (iii) and (iv).

Although the components may be admixed in any order, it is generallypreferable to combine the components in the following order: combinedcell surface receptor-binding/polycationic DNA-binding or RNA-bindingcomponent; DNA or RNA component, for example, in the order:polyethylenimine-peptide component; DNA or RNA component.

A transfection mixture comprising a cell surface receptor-bindingcomponent and a polycationic nucleic acid-binding component may be usedto produce a nucleic acid-containing transfection vector complex asdescribed above by the incorporation of a nucleic acid with the mixture,for example, by admixture. Alternatively, the transfection mixture maybe used for the production of a vector complex which comprises, insteadof the nucleic acid component, any other component that is capable ofbinding to the polycationic nucleic-acid binding component, for example,a protein.

The individual components of a transfection mixture of the invention areeach as described above in relation to the transfection vector complex.The preferred components, preferred combinations of components,preferred ratios of components and preferred order of mixing, both withregard to the mixture and to the production of a vector complex, are asdescribed above in relation to the transfection vector complex.

The present invention also provides a process for expressing a nucleicacid in host cells, which comprises contacting the host cells in vitroor in vivo with a receptor-targeted vector complex of the inventioncomprising the nucleic acid and then culturing the host cells underconditions that enable the cells to express the nucleic acid.

The present invention further provides a process for the production of aprotein in host cells, which comprises contacting the host cells invitro or in vivo with a receptor-targeted vector complex of theinvention that comprises a nucleic acid that encodes the protein,allowing the cells to express the protein, and obtaining the protein.The protein may be obtained either from the host cell or from theculture medium.

The present invention further provides a method of transfecting cellscomprising subjecting the cells to a vector complex according to theinvention.

The invention further provides cells, transfected with a nucleic acid bya method according to the invention, and also the progeny of such cells.

The present invention further provides a disease model for use intesting candidate pharmaceutical agent, which comprises cellstransfected by a method according to the invention with a nucleic acidsuitable for creating the disease model.

The present invention also provides a pharmaceutical composition whichcomprises a receptor-targeted vector complex of the invention comprisinga nucleic acid in admixture or conjunction with a pharmaceuticallysuitable carrier. The composition may be a vaccine.

The present invention also provides a method for the treatment orprophylaxis of a condition caused in a human or in a non-human animal bya defect and/or a deficiency in a gene, which comprises administering tothe human or to the non-human animal a receptor-targeted vector complexof the invention comprising a nucleic acid suitable for correcting thedefect or deficiency.

The present invention also provides a method for therapeutic orprophylactic immunisation of a human or of a non-human animal, whichcomprises administering to the human or to the non-human animal areceptor-targeted vector complex of the invention comprising anappropriate nucleic acid.

The present invention also provides a method of anti-sense therapy of ahuman or of a non-human animal, comprising anti-sense DNA administeringto the human or to the non-human animal a receptor-targeted vectorcomplex of the invention comprising the anti-sense nucleic acid.

The present invention also provides the use of a receptor-targetedvector complex of the invention comprising a nucleic acid for themanufacture of a medicament for the prophylaxis of a condition caused ina human or in a non-human animal by a defect and/or a deficiency in agene, for therapeutic or prophylactic immunisation of a human or of anon-human animal, or for anti-sense therapy of a human or of a non-humananimal.

A non-human animal is, for example, a mammal, bird or fish, and isparticularly a commercially reared animal.

The nucleic acid, either DNA or RNA, in the vector complex isappropriate for the intended use, for example, for gene therapy, genevaccination, or anti-sense therapy. The DNA or RNA and hence the vectorcomplex is administered in an amount effective for the intended purpose.

The treatments and uses described above may be carried out byadministering the respective vector complex, agent or medicament in anappropriate manner, for example, administration may be topical, forexample, in the case of airway epithelia.

In a further embodiment, the present invention provides a kit comprisinga receptor-targeted vector complex of the invention comprising a nucleicacid.

The present invention also provides a kit that comprises the followingitems: (a) a cell surface receptor-binding component; (b) a polycationicnucleic acid-binding component, and (c) a lipid component. Such a kitmay further comprise (d) a nucleic acid. Such a nucleic acid may besingle-stranded or double stranded and may be a plasmid or an artificialchromosome. The nucleic acid component may be provided by a vectorcomplex suitable for the expression of the nucleic acid, the vectorcomplex being either empty or comprising the nucleic acid. For in vitropurposes, the nucleic acid may be a reporter gene. For in vivo treatmentpurposes, the nucleic acid may comprise DNA appropriate for thecorrection or supplementation being carried out. Such DNA may be a gene,including any suitable control elements, or it may be a nucleic acidwith homologous recombination sequences. It has been found thatpeptide/DNA/lipid/polycationic nucleic acid-binding component complexesare especially stable in salt free buffer (for example in water, or 5%dextrose).

The present invention also provides a kit that comprises the followingitems: (a) a cell surface receptor-binding component; and (b) apolycationic nucleic acid-binding component. Such a kit may furthercomprise (d) a nucleic acid. Such a nucleic acid may be single-strandedor double stranded and may be a plasmid or an artificial chromosome. Thenucleic acid component may be provided by a vector complex suitable forthe expression of the nucleic acid, the vector complex being eitherempty or comprising the nucleic acid. The nucleic acid component may beprovided by a vector complex suitable for the expression of the nucleicacid, the vector complex being either empty or comprising the nucleicacid. For in vitro purposes, the nucleic acid may be a reporter gene.For in vivo treatment purposes, the nucleic acid may comprise DNAappropriate for the correction or supplementation being carried out.Such DNA may be a gene, including any suitable control elements, or itmay be a nucleic acid with homologous recombination sequences. It hasbeen found that peptide/DNA/polycationic nucleic acid-binding componentcomplexes are especially stable in salt free buffer (for example inwater, or 5% dextrose).

The components (a) to (d) kit are, for example, as described above inrelation to a cell surface receptor-targeted transfection vector complexor a mixture as described above.

A kit generally comprises instructions, which preferably indicate thepreferred ratios of the components and the preferred order of use oradmixing of the components, for example, as described above. A kit maybe used for gene therapy, gene vaccination or anti-sense therapy.Alternatively, it may be used for transfecting a host cell with anucleic acid encoding a commercially useful protein i.e. to produce aso-called “cell factory”.

In a kit of the invention the components including the preferredcomponents are, for example, as described above in relation to a vectorcomplex of the present invention.

The polycationic nucleic acid binding component is preferably anoligolysine, as described above. The lipid component is preferablycapable of forming a cationic liposome, and preferably is or comprisesDOPE and/or DOTMA, for example, an equimolar mixture thereof, or is orcomprises DOSPA, for example, a mixture of DOPE and DOSPA, for examplein the weight ratio DOPE:DOSPA of 1:3. The rations between thecomponents are preferably as described above, as is the order of mixingof the components.

Targets for gene therapy are well known and include monogenic disorders,for example, cystic fibrosis, various cancers, and infections, forexample, viral infections, for example, with HIV. For example,transfection with the p53 gene offers great potential for cancertreatment. Targets for gene vaccination are also well known, and includevaccination against pathogens for which vaccines derived from naturalsources are too dangerous for human use and recombinant vaccines are notalways effective, for example, hepatitis B virus, HIV, HCV and herpessimplex virus. Targets for anti-sense therapy are also known. Furthertargets for gene therapy and anti-sense therapy are being proposed asknowledge of the genetic basis of disease increases, as are furthertargets for gene vaccination. The present invention enhances thetransfection efficiency and hence the effectiveness of the treatment.

Vector complexes of the invention may be effective for intracellulartransport of very large DNA molecules, for example, DNA larger than 125kb, which is particularly difficult using conventional vectors. Thisenables the introduction of artificial chromosomes into cells.

Transfection of the airways, for example, the bronchial epitheliumdemonstrates utility for gene therapy of, for example, respiratorydiseases, such as cystic fibrosis, emphysema, asthma, pulmonoryfibrosis, pulmonary hypertension and lung cancer.

Cystic fibrosis (CF) is the most common monogenic disorder in theCaucasian population. Morbidity is mainly associated with lung disease.CF is caused by mutations in the gene encoding the cystic fibrosistransmembrane conductance regulator protein (CFTR), a cell membranechannel that mediates secretion of chloride ions. Correction of thisdefect in the bronchial cells by CFTR gene transfer will correct thebiochemical transport defect and, hence, the lung disease. Clinicaltrials so far have generated encouraging data but highlighted the needfor more efficient, non-toxic vectors.

The enhanced levels of transfection make the method of the inventionparticularly suitable for the production of host cells capable ofproducing a desired protein, so-called “cell fac-tories”. For long-termproduction, it is desirable that the introduced nucleic acid isincorporated in the genome of the host cell, or otherwise stablymaintained. That can be readily ascertained. As indicated above, therange of proteins produced in this way is large, including enzymes forscientific and industrial use, proteins for use in therapy andprophylaxis, immunogens for use in vaccines and antigens for use indiagnosis.

Accordingly, the present invention provides a method of testing drugs ina tissue model for a disease, wherein the tissue model comprisestransgenic cells obtained by transfecting cells with a nucleic acid bycontacting the cell with a receptor-targeted vector complex of theinvention comprising a nucleic acid.

The present invention is especially useful with a receptor targetedvector complex that is capable of high efficiency transfection. In apreferred embodiment, the vector complex comprises four modularelements; an oligolysine, especially [K]₁₆, DNA-binding or RNA-bindingelement; a high affinity cell surface receptor-binding peptide, forexample, a peptide described herein; a DNA or RNA sequence, optionallyin a plasmid, and optionally regulated by a viral promoter and anenhancing element; the cationic liposome DOTMA/DOPE (lipofectin). Thecombination of oligolysine-peptide/DNA or RNA complex with the cationicliposome formulation DOTMA/DOPE is a potent combination. Alternatively aDOPE/DOSPA formulation may be used instead of or in addition to aDOTMA/DOPE formulation. The optimisation of variables associated withcomplex formation and the mode of transfection by LID vector complexeshas been demonstrated.

The most important variables in the formation of optimal LIDtransfection complexes appear to be the ratio of the three componentsand their order of mixing.

The invention further provides a method for identifying a cell surfacereceptor binding ligand for use in a non-viral transfection vectorcomplex comprising the steps:

-   -   a) selecting phage from a phage peptide library according to        their binding affinity to cells of interest by bringing the        phage into contact with the cells of interest and washing away        non-binding phage and then extracting bound phage particles,    -   b) repeating step (a) if necessary, and preferably    -   c) selecting from the phage obtained in steps a) and b) those        phage which bind to the cell of interest with high affinity        using a whole cell ELISA.

Preferably, the stringency of the wash in step a) is increased after thefirst round of selection by washing at low pH by washing multiple times.

BRIEF DESCRIPTION OF THE DRAWINGS

The following non-limiting Examples illustrate the present invention.The Examples refer to the accompanying drawings, in which:

FIG. 1 shows the enhancement of phage binding to HAE cells in successiverounds of selection for the C7C library and the 12 mer peptide librarystarting materials.

FIG. 2 shows the binding specificity of individual phage clones in awhole cell ELISA assay. Binding affinity to HAE cells, to 3T3 controlcells and to the ELISA plate are shown.

FIG. 3 shows the relative efficiency of transfection of HAE cellsachieved by transfection complexes according to the invention.

FIG. 4 shows the relative efficiency of transfection of HAE cellsachieved by transfection complexes according to the invention andscrambled control peptides.

FIG. 5 shows the relative efficiency of transfection of Neuro-2A cellsachieved by transfection complexes according to the invention and acontrol peptide, peptide 6.

FIG. 6 shows the relative efficiency of transfection of IMR32 cellsachieved by transfection complexes according to the invention and acontrol peptide, peptide 6.

FIG. 7 shows the relative efficiency of transfection of rabbitadventitial cells achieved by transfection complexes according to theinvention and a control peptide, peptide 6.

FIG. 8 shows the relative efficiency of transfection of 3T3 cellsachieved by transfection complexes according to the invention and acontrol peptide, peptide 6.

EXAMPLES Materials & Methods Example 1

Peptide Library

The peptide library used in this study, C7C, was obtained from NewEngland Biolabs Inc. Phage growth, titration and amplificationprocedures were performed as described in the manufacturer's handbook.The library consisted of random peptide sequences seven residues inlength and flanked by cystine residues to allow cyclisation by oxidationin air. The library is likely to contain at least 1×10⁹ different aminoacid sequences.

Selection of Phage from the Library

HAE cells were grown to confluence in 24-well plates. The HAE cells usedwere 1HAEo− cells obtained as a gift from Dr. Dieter Gruenert of theUniversity of California, San Francisco (now of the University ofVermont). Cells were washed twice in Tris-buffered saline, pH 7.4 (TBS)before blocking cells with 2 ml 2% Marvel, 5% bovine serum albumin(BSA)-TBS per well for 30 minutes at 4° C. The blocker was removed and2×10¹¹ phage were added in 1 ml of 2% Marvel, 5% BAS-TBS. The phage wereallowed to bind for 2 hours with shaking at 4° C. before washing fiveitems with 2% BSA-TBS and 5 minutes shaking at 4° C. followed by anotherfive washes with 2% BSA-TBS for a few seconds only. Phage were eluted bythe addition of 400 μl 76 mM citrate buffer pH 2.5 to the wells for 10minutes with shaking at 4° C. The eluate was removed and neutralisedwith 600 μl 1M Tris buffer pH 7.5 and retained as the eluted fraction.The remaining cells were lysed with 1 ml 30 mM Tris buffer pH 8.0, 1 mMEDTA for 1 hour on ice. The cells were scraped from the plate, theeluate transferred to a microcentrifuge tube, and vortexed briefly. Thateluate was retained as the cell-associated fraction.

The above described process was repeated three times. In the second andthird rounds, the stringency of selection was increased by introductionof preselection steps to remove phage that bind to the plastic or tocomponents in the medium and by increasing the number of washesfollowing phage binding. The number of phage present in each eluate (inplaque forming units, PFU) is shown in FIG. 1.

Whole Cell HAE Cell Binding ELISA

Binding of phage to tissue cultured HAE cells was investigated by wholecell ELISA. Approx. 8×10⁴ HAE cells in 100 ml Hanks Balanced SaltsSolution (HBSS) were added to each well of a 96 well plate and incubatedat 37° C. until cells had adhered. The cells were washed gently in HBSSbefore blocking by the addition of 0.5% BSA in HBSS for 30 mins. 1×10¹⁰phage particles in blocker solution were added to each well and allowedto bind at room temperature for 40 minutes. Unbound phage were removedby washing twice with HBSS, and bound phage were fixed to the cells byincubation in 3.7% paraformaldehyde for 10 mins. Cells were washed inPBS and incubated in blocking buffer for 45 mins, followed by threewashes in PBS. Bound phage were detected by the addition of horseradishperoxidase (HRP)-conjugated anti-M13 antibody diluted 1:5000 in blockingbuffer for 1 hour, before washing three times in PBS and developing theELISA with 2,2′-azino-bis(3-ethylbenzthiazoline 6-sulfonic acid) (ABTS)substrate solution and reading the absorbance on a plate-readingspectrophotometer at 405 nm. The experiment was repeated using 3T3 cellsand using empty wells and the comparison of binding affinities enabledthe identification of phage that bound selectively to HAE cells. Theresults for selected peptides are shown in FIG. 2.

Peptide-encoding DNA of 12 phage clones that displayed high HAE cellavidity and specificity were sequenced and the peptide sequence deduced.The sequences deduced are shown in table 1. Three major peptide motifs,KSM/RSM, LXHK (SEQ ID NO: 2) and LXHKSMP (SEQ ID NO: 18) were identifiedamongst the 12 sequences and one sequence, PSGAARA (SEQ ID NO: 3) thatcontained none of the other three motifs. The sequences were comparedand ranked for their binding strength by ELISA using a range of phagetitres (table 3). It was found that the sequence LPHKSMP (SEQ ID NO: 5)(peptide P) had the highest binding affinity. This sequence and theclosely related peptide LQHKSMP (SEQ ID NO: 4) (peptide Q) and acontrol, scrambled version of peptide P were selected for transfectionexperiments.

Peptide Synthesis

The following oligolysine-peptides were prepared for transfectionexperiments:

Peptide P: [K]₁₆-GACLPHKSMPCG - binds to HAE cells Peptide Q:[K]₁₆-GACLQHKSMPCG - binds to HAE cells Peptide S: [K]₁₆-GACYKHPGFLCG -non-binding control Peptide 12: [K]₁₆-XSXGACRRETAWACG - binds to alpha 5beta 1 integrins (X = ε-amino hexanoic acid) K16: DNA binding moiety, notargeting ligand.

The oligolysine-peptides were synthesised using standard solid phaseoligopeptide synthesis methods.

Transfection Experiments

Peptides identified from phage that displayed desirable cell bindingcharacteristics were synthesised using standard solid-phase peptidesynthetic chemistry and a sixteen-lysine tail was attached usingstandard synthesis methods. Control peptide (S), consisted of the sameamino acid constituents as the targeting peptide P but in a randomisedorder, was synthesised for incorporation into lipopolyplex formulations.Transfections of HAE and 3T3 cells were performed in 96 well platedcontaining 20,000 cells plated 24 h earlier. In the transfectioncomplex, peptide to DNA charge ratios (+/−) were used at 1.5:1, 3:1 and7:1. The lipid component was maintained at a constant proportion, byweight, relative to DNA of 0.75:1. Prior to making transfectioncomplexes the lipid component was diluted to a concentration of 15 μgper ml, the peptide was prepared at 0.1 mg/ml and the DNA was at 20 μgper ml. All dilutions were performed with OptiMEM reduced serum tissueculture medium (Life Technologies). Transfection complexes were made bymixing of components in the order 1) lipid then 2) peptide and finally3) DNA, then diluted with OptiMEM to a concentration relative to the DNAcomponent of 0.25 μg DNA per 200 μl which volume was added to each well.Each group was performed in replicates of six. The vector complexsuspension was then applied to cells within 5 minutes of preparation.Transfection incubations were performed at 37° C. for 4 h. Luciferasereporter gene assays in cell free extracts were performed after 48 hincubation using a kit from Promega according to the manufacturer'sprotocol. Light units were standardised to the protein concentrationwithin each extract. The results of the transfection experiments areshown in FIG. 3.

At a 7:1 charge ratio the transfection efficiency of complexescontaining peptide P was five fold-higher than the next best peptide,peptide 12 at a 3:1 ratio. Peptide P was more than 150-fold better thanPeptide S at the charge ratio of 7:1 indicating that the transfectionefficiency was receptor specific. Complexes containing peptide P werealmost nine-fold higher than K₁₆, again indicating receptor specificity.This result also suggests that peptide S is less than a tenth as good intransfection complexes as peptide K₁₆. This may be explained by sterichindrance by the scrambled motif in peptide S.

The difference in transfection performance between peptides P and Q wasunexpected as peptide Q (LQHKSMP) (SEQ ID NO: 4) varies from peptide Pby a single amino acid residue. This result suggests that bindingproperties alone are not sufficient to explain the transfectionpotential of the peptides. These results also suggest that the LIDvector complex system may be retargeted to other specific peptidesdescribed herein and may be useful for targeted gene delivery toepithelial cells in vivo or in vitro.

Example 2

Example 2 is a similar series of experiment to Example 1, withrelatively minor changes in a number of conditions.

Cell Lines

The human airway epithelial cell line (HAEo−) was maintained in Eagle'sminimal essential medium (MEM) HEPES modification (Sigma, Poole)containing 10% foetal calf serum (FCS), penicillin and streptomycin, andL-glutamine. The mouse fibroblast cell line 3T3 and the humanneuroblastoma cell line IMR32 were grown in Dulbecco's MEM withGlutamax-1, without sodium pyruvate, with 4500 mg/L glucose, withpyridoxine (Gibco BRL) with 10% FCS, penicillin and streptomycin added.Neuro-2A cells were maintained in Dulbecco's MEM with Glutamax-1 (GibcoBRL) with 10% FCS, sodium pyruvate, penicillin and streptomycin andnon-essential amino acids.

Panning Cells in Monolayer

HAEo− cells were grown to confluence in 24 well plates. Cells werewashed twice in TBS before blocking cells with 2 mls 2% Marvel, 5%BSA-TBS per well for 30 mins at 4° C. The blocker was removed and 2×10¹¹phage were added in 1 ml of 2% Marvel, 5% BSA-TBS. The phage wereallowed to bind for 2 hours shaking at 4° C. before washing five timeswith 2% BSA-TBS for 5 mins shaking at 4° C., followed by another fivewashes with 2% BSA-TBS for a few seconds only. Phage were eluted by theaddition of 400 l 76 mM citrate buffer pH 2.5 to the wells for 10 minsshaking at 4° C. The eluate was removed and the remaining cells werelysed with 1 ml 30 mM Tris pH 8.0, 1 mM EDTA for 1 hour on ice. Thecells were scraped from the plate, the eluate transferred to aneppendorf, and vortexed briefly. This eluate was saved as thecell-associated fraction. The phage from this elution were titrated asplaque forming units (PFU) as described in the literature supplied withthe library by NEB, before amplification of the phage in E. coli ER2738cells as described in the literature. For the second round of panning,2×10¹¹ of the amplified phage from the previous round was used as theinput phage. However, in order to reduce the number of plastic andblocking molecule-binding phage isolated, four pre-selection steps ofadding the phage to a blocked well with no cells for 30 mins at 4° C.was carried out before adding the phage to the HAEo− cells. Thestringency of washing as also increased in both the second and thirdrounds by the addition of a 10 min wash at 4° C. using 1 ml 76 mMcitrate buffer pH3.5. For the third round, 2×10¹¹ amplified phage fromthe second round was preselected in 5 blocked wells containing no cellsfor 30 mins each, followed by 1 well for 1 hour at 4° C. Phage bindingand elution was as described for the second round. Following titrationof the third round eluate, single well isolated plaques were picked,amplified and purified for sequencing and clone binding characterisationby whole cell ELISA.

Phage Sequencing

The phage were purified from small scale PEG preps (see suppliersmethods) and single stranded phage DNA was prepared for sequencing usingthe method described in Phage display of Peptides and Proteins Edited byBrian K. Kay, Jill Winter and John McCafferty. Briefly, the protein coatwas removed from the sample by phenol chloroform extraction, and the DNApelleted by ethanol precipitation. Trace salt was washed from the pelletwith ice cold 70% ethanol before resuspending the DNA in TE.

Between 50 and 100 ng purified DNA was used in a Big Dye terminatorcycle sequencing reaction (ABI) using the −96 primer supplied with thelibrary and purified for loading by ethanol precipitation as describedin Big Dye kit instructions. The samples were run on an ABI 377sequencer and the results analysed using the Vector NTI program.

Whole Cell ELISA

Approx. 8×10⁴ HAE cells in 100 ml HBSS were added to each well of a 96well plate and incubated at 37° C. until cells had adhered. The cellswere washed gently in HBSS before blocking by the addition of 0.5% BSAin HBSS for 30 mins. 1×10¹⁰ phage particles in blocker were added toeach well and allowed to bind at room temperature for 40 mins. Unboundphage were removed by washing twice with HBSS, and bound phage werefixed to the cells by incubation in 3.7% paraformaldehyde for 10 mins.Cells were washed in PBS and incubated in blocking buffer for 45 mins,followed by three washes in PBS. Bound phage were detected by theaddition of HRP-conjugated anti-M13 antibody diluted 1:5000 in blockingbuffer for 1 hour, before washing three times in PBS, developing theELISA with ABTS solution, and reading the absorbance at 405 nm.

Peptide Synthesis

The [K]16—forms of the cyclised peptides (as shown in Table 6) weresynthesised by standard solid phase synthesis by Alta Biosciences,Birmingham, and the Department of Chemistry, UCL.

TABLE 6 Phage Peptide Peptide peptide SEQ.ID. name synthesised SEQ.ID.LPHKSMP 5 P [K]₁₆-GACLPHKSMPCG 13 LQHKSMP 4 Q [K]₁₆-GACLQHKSMPCG 12YGLPHKF 19 Y [K]₁₆-GACYGLPHKFCG 44 SERSMNF 7 E [K]₁₆-GACSERSMNFCG 27VKSMVTH 6 V [K]₁₆-GACVKSMVTHCG 28 PSGAARA 3 S [K]₁₆-GACPSGAARACG 29YKHPGFL 21 S/YS [K]₁₆-GACYKHPGFLCG 30 NSFMESR 22 ES [K]₁₆-GACNSFMESRCG31 ASSARPA 23 OS [K]₁₆-GACAGSARPACG 32 PLSHQMK 24 QS [K]₁₆-GACPLSHQMKCG33 HPPMSKL 25 PS [K]₁₆-GACHPPMSKLCG 34 RRETEWA 26 6 [K]₁₆-GACRRETEWACG35

For the avoidance of doubt, all of the sequences in Table 5 form part ofthe present invention.

Transfections

Lipopolyplex Formation

Complexes were allowed to form electrostatically in a tube by adding thefollowing components in the following order. 50 μl of Lipofectin (LifeTechnologies Ltd) diluted to a concentration of 30 μg/ml in OptiMEM,followed by 70 μl peptide (at varying concentrations in OptiMEM foroptimisation of the peptide:DNA charge ratio in the complex), with 50 μlof the luciferase reporter plasmid pCILuc at a concentration of 40 ug/mlin Optimem added finally. The complex was mixed by pipetting brieflybefore diluting in Optimem to a final volume of 1.57 mls.

Transfection

The media was removed from subconfluent HAEo− cells plated at 2×10⁴cells/well overnight in 96 well plates and 200 μl of complex (approx.0.25 μg of plasmid DNA) added to each well, leaving minimal time betweenpreparing the complex and adding to the cells. All transfections werecarried out in 6 wells each. The cells were incubated with the complexesfor 4 hours before replacing with normal media for 48 hours, after whichreporter gene expression was analysed by luciferase assay (Promega).

Luciferase Assay

The cells were rinsed twice with PBS before the addition of 100 μl ofreporter lysis buffer (Promega, diluted 1 in 5 in dH₂O) to the cells for20 mins at 4° C. before freeze-thawing. 20 μl of the lysate wastransferred to a white plate and the luciferase was measured by a Lucy1luminometer following the addition of 100 μl of reagent.

The protein present in each transfection well was calculated using theBio-Rad protein assay reagent (based on the Bradford assay), adding 20μl from the luciferase test to 200 μl of the reagent diluted 1 in 5,incubating for 10 mins at room temperature and reading the absorbance at590 nm. The total protein present per well was calculated fromcomparison with a range of BSA standards.

The results of the transfection experiments are shown in FIG. 4.Transfection of HAEo− cells with phage derived peptides and theirscrambled controls was carried out with a range of peptide:DNA chargeratios including 1.5:1, 3:1 and 7:1. The ratio giving the highesttransfection efficiency (determined as RLU/mg) for each peptide is shownin the figure. Controls include cells with no transfection complexesadded (OptiMEM only) and peptide 6, an integrin binding peptide. Eachresult is the mean of 6 values and error bars represent the standarddeviation about the mean.

Example 3

The transfection experiments described above were repeated usingNeuro-2A cells, IMR32 cells, rabbit adventitial fibroblast cells and 3T3cells. For analysis of transfections of those cell lines, cells wereplated to subconfluence overnight before transfecting in the same manneras above, analysing reporter gene expression after 24 hours. The resultsare shown in FIGS. 5 to 8.

Transfection of Neuro-2A cells with phage-derived peptides was carriedout with a range of peptide:DNA charge ratios including 1.5:1, 3:1 and7:1. The ratio giving the highest transfection efficiency (determined asRLU/mg) for each peptide is shown in FIG. 5. Controls included cellswith no transfection complexes added (OptiMEM only) peptide 6, anintegrin binding peptide, and peptide S, the scrambled version ofpeptide Y. Each result is the mean of 6 values and error bars representthe standard deviation about the mean.

Transfection of IMR32 cells with phage-derived peptides was carried outwith a range of peptide:DNA charge ratios including 1.5:1, 3:1 and 7:1.The ratio giving the highest transfection efficiency (determined asRLU/mg) for each peptide is shown in FIG. 6. Controls include cells withno transfection complexes added (OptiMEM only) peptide 6, an integrinbinding peptide, and peptide S, the scrambled version of peptide Y. Eachresult is the mean of 6 values and error bars represent the standarddeviation about the mean.

Transfection of rabbit adventitial fibroblast cells with phage-derivedpeptides was carried out with a range of peptide:DNA charge ratiosincluding 1.5:1, 3:1 and 7:1. The ratio giving the highest transfectionefficiency (determined as RLU/mg) for each peptide is shown FIG. 7.Controls include cells with no transfection complexes added (OptiMEMonly) peptide 6, an integrin binding peptide, and peptide S, thescrambled version of peptide Y. Each result is the mean of 6 values anderror bars represent the standard deviation about the mean.

Transfection of 3T3 cells with phage-derived peptides was carried outwith a range of peptide:DNA charge ratios including 1.5:1, 3:1 and 7:1.The ratio giving the highest transfection efficiency (determined asRLU/mg) for each peptide is shown in FIG. 8. Controls include cells withno transfection complexes added (OptiMEM only) peptide 6, an integrinbinding peptide, and peptide S, the scrambled version of peptide Y. Eachresult is the mean of 6 values and error bars represent the standarddeviation about the mean.

It is seen in FIGS. 5, 6 and 7 that transfection of Neuro-2A cells,IMR32 cells, rabbit adventitial fibroblast cells with the peptides wasof similar efficiency or lower than transfection with peptide 6. Only in3T3 cells (FIG. 8) was the transfection efficiency above that seen withpeptide 6, with peptides Q and E showing efficiencies of approximately1.5 times that seen with peptide 6. All transfections showedefficiencies above that of the scrambled peptide. These results maysuggest that the molecules bound by the peptide are present on othercell types and in other species but maybe in altered forms or atdifferent densities compared to HAEo− cells.

REFERENCES

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1. An isolated peptide of 7 to 100 amino acids comprising an amino acidsequence including KSM , wherein said amino acid sequence is selectedfrom LX⁵HKSMP [SEQ ID NO.:18], in which X⁵ is P or Q, and VKSMVTH [SEQID NO.:6], and wherein said amino acid sequence binds to a cell surfacereceptor.
 2. The isolated peptide according to claim 1, wherein theamino acid sequence is LQHKSMP [SEQ ID NO:4] or LPHKSMP [SEQ ID NO:5].3. The isolated peptide according to claim 1, consisting of 7 to 20amino acids.
 4. The isolated peptide according to claim 1, consisting of7 to 12 amino acids.
 5. The isolated peptide according to claim 1,wherein the peptide is comprised within a cyclic region of amino acids.6. The isolated peptide according to claim 5, wherein the peptidecomprises two or more cysteine residues capable of forming one or moredisulphide bond(s).
 7. The isolated peptide according to claim 1,wherein the peptide is linked to a polycationic nucleic acid-bindingcomponent.
 8. The isolated peptide according to claim 7, wherein thepolycationic nucleic acid-binding component is selected frompolyethylenimine and an oligo-lysine molecule consisting of from 5 to 25lysine moieties.
 9. The isolated peptide according to claim 7, whereinthe peptide is linked to the polycationic nucleic acid-binding componentvia a spacer element.
 10. The isolated peptide according to claim 9,wherein the spacer element is GG or GA or is longer and/or morehydrophobic than the dipeptide spacers GG (glycine-glycine) and GA(glycine-alanine).
 11. A peptide derivative of formula A-B-C wherein Ais a polycationic nucleic acid-binding component, B is a spacer element,and C is a peptide according to claim
 1. 12. A mixture comprising a cellsurface receptor-binding component, a polycationic nucleic acid-bindingcomponent, and optimally a lipid component, the cell surfacereceptor-binding component being a peptide as defined in claim
 1. 13.The mixture according to claim 12, wherein said cell surfacereceptor-binding component is a peptide of 7 to 20 amino acidscomprising an amino acid sequence selected from the group consisting of:VKSMVTH [SEQ ID NO.:6], LQHKSMP [SEQ ID NO:4] or LPHKSMP [SEQ ID NO:5],and cyclic peptides thereof.
 14. The mixture according to claim 13,wherein the polycationic nucleic acid-binding component is comprised of3 to 100 cationic monomers and is selected from the group consisting ofoligolysine, polyethyleneimine, and combinations thereof.
 15. Themixture according to claim 13 or claim 14, wherein the lipid componentis selected from the group consisting of dioleylphosphatidyl-ethanolamine (DOPE),N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),or combinations thereof.
 16. The mixture according to claim 14, whichcomprises an equimolar mixture of DOPE and DOTMA as the lipid component,and [K]₁₆ as the polycationic component nucleic acid-binding component.17. A process for producing a nucleic acid-containing transfectionvector complex, which comprises incorporating a nucleic acid with amixture according to claim
 12. 18. A kit that comprises (i) nucleicacid, (ii) optionally, a lipid component, (iii) a polycationic nucleicacid-binding component, and (iv) a cell surface receptor bindingcomponent, comprising a peptide as defined in claim
 1. 19. A non-viraltransfection complex comprising: (i) a nucleic acid, (ii) optionally, alipid component, (iii) a polycationic nucleic acid-binding component,and (iv) a cell surface receptor binding component, comprising a peptideas defined in claim
 1. 20. A method of transfecting a cell with anucleic acid, which comprises contacting the cell in vitro or in vivowith a complex according to claim
 19. 21. A pharmaceutical compositionwhich comprises a complex according to claim 19, in admixture orconjunction with a pharmaceutically suitable carrier.
 22. A method forexpressing a gene in a human or in a non-human animal with a defectand/or a deficiency in a gene, which comprises administering a complexas defined in claim 19 to the human or to the non-human animal.
 23. Amethod for inducing an immune response in a human or a non-human animal,which comprises administering a complex as defined in claim 19 to thehuman or to the non-human animal.
 24. A method of inhibiting theexpression of a gene, which comprises administering a complex as definedin claim 19 to a human or to a non-human animal, wherein the expressionof the gene is inhibited by the expression of an antisense nucleic acid.